Abstract
Ion-exchange chromatography (IEC) allows for the separation of ionizable molecules on the basis of differences in charge properties. Its large sample-handling capacity, broad applicability (particularly to proteins and enzymes), moderate cost, powerful resolving ability, and ease of scale-up and automation have led to it becoming one of the most versatile and widely used of all liquid chromatography (LC) techniques. In this chapter, we review the basic principles of IEC, as well as the broader criteria for selecting IEC conditions. By way of further illustration, we outline protocols necessary to partially purify a serine peptidase from bovine whole brain cytosolic fraction, covering crude tissue extract preparation through to partial purification of the target enzyme using anion-exchange chromatography. Protocols for assaying total protein and enzyme activity in both pre- and post-IEC fractions are also described. The target serine peptidase, prolyl oligopeptidase (POP, EC3.4.21.26), is an 80-kDa enzyme with endopeptidase activity towards peptide substrates of ≤30 amino acids. POP is a ubiquitous post-proline cleaving enzyme with particularly high expression levels in the mammalian brain, where it participates in the metabolism of neuroactive peptides and peptide-like hormones (e.g. thyroliberin, gonadotropin-releasing hormone).
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References
Fritz, J. S. (2004) Early milestones in the development of ion-exchange chromatography: a personal account. J Chromatogr A 1039, 3–12.
Lucy, C. A. (2003) Evolution of ion-exchange: from Moses to the Manhattan Project to modern times. J Chromatogr A 1000, 711–24.
Kent, U. M. (1999) Purification of antibodies using ion-exchange chromatography. Methods Mol Biol 115, 19–22.
Levison, P. R. (2003) Large-scale ion-exchange column chromatography of proteins: comparison of different formats. J Chromatogr B Analyt Technol Biomed Life Sci 790, 17–33.
Cummins, P. M. and O’Connor, B. (1996) Bovine brain pyroglutamyl aminopeptidase (type-I): purification and characterization of a neuropeptide-inactivating peptidase. Int J Biochem Cell Biol 28, 883–93.
Wu, B., Wu, L., Chen, D., Yang, Z., and Luo, M. (2009) Purification and characterization of a novel fibrinolytic protease from Fusarium sp. CPCC 480097. J Ind Microbiol Biotechnol 36, 451–59.
Knudsen, H. L., Fahrner, R. L., Xu, Y, Norling, L. A., and Blank, G. S. (2001) Membrane ion-exchange chromatography for process-scale antibody purification. J Chromatogr A 907, 145–54.
Mant, C. T. and Hodges, R. S. (2008) Mixed-mode hydrophilic interaction/cation-exchange chromatography: separation of complex mixtures of peptides of varying charge and hydrophobicity. J Sep Sci 31, 1573–84.
Fekkes, D., Voskuilen-Kooyman, A., Jankie, R., and Huijmans, J. (2000) Precise analysis of primary amino acids in urine by an automated high-performance liquid chromatography method: comparison with ion-exchange chromatography. J Chromatogr B Biomed Sci Appl 744, 183–88.
Yang, Y., Hebron, H. R., and Hang, J. (2008) High performance DNA purification using a novel ion-exchange matrix. J Biomol Tech 19, 205–10.
Bonn, G. (1987) High-performance liquid chromatographic isolation of 14C-labelled gluco-oligosaccharides, monosaccharides and sugar degradation products on ion-exchange resins. J Chromatogr 387, 393–98.
Hajós, P. and Nagy, L. (1998) Retention behaviours and separation of carboxylic acids by ion-exchange chromatography. J Chromatogr B Biomed Sci Appl 717, 27–38.
Ahmed, H. (ed.) (2004) Principals and reactions of protein extraction, purification and characterization. CRC Press, Boca Raton, FL.
Männisto, P. T., Venäläinen, J., Jalkanen, A., and García-Horsman, J. A. (2007) Prolyl oligopeptidase: a potential target for the treatment of cognitive disorders. Drug News Perspect 20, 293–305.
Polgár, L. (2002) The prolyl oligopeptidase family. Cell Mol Life Sci 59, 349–62.
Kalwant, S. and Porter, A. G. (1991) Purification and characterization of human brain prolyl endopeptidase. Biochem J 276, 237–44.
Sharma, K. K. and Ortwerth, B. J. (1994) Purification and characterization of prolyl oligopeptidase from bovine lens. Exp Eye Res 59, 107–15.
Cunningham, D. F. and O’Connor, B. (1998) A study of prolyl endopeptidase in bovine serum and its relevance to the tissue enzyme. Int J Biochem Cell Biol 30, 99–114.
Birney, Y. A. and O’Connor, B. F. (2001) Purification and characterization of a Z-pro-prolinal-insensitive Z-Gly-Pro-7-amino-4-methyl coumarin-hydrolyzing peptidase from bovine serum: a new proline-specific peptidase. Protein Expr Purif 22, 286–98.
Cunningham, D. F. and O’Connor, B. (1996) Identification and initial characterization of a N-benyloxycarbonyl-prolyl-prolinal (Z-Pro-Prolinal)-insensitive 7-(N-benzyloxycarbonyl-glycyl-prolyl-amido)-4-methylcoumarin (Z-Gly-Pro-NH-Mec)-hydrolyzing peptidase in bovine serum. Eur J Biochem 244, 900–3.
Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal Biochem 72, 248–54.
Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., et al. (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150, 76–85.
Yoshimoto, T., Ogita, K., Walter, R., Koida, M., and Tsuru, D. (1979) Post-proline cleaving enzyme: synthesis of a new fluorogenic substrate and distribution of the endopeptidase in rat tissues and body fluids of man. Biochim Biophys Acta 569, 184–92.
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Cummins, P.M., Dowling, O., O’Connor, B.F. (2011). Ion-Exchange Chromatography: Basic Principles and Application to the Partial Purification of Soluble Mammalian Prolyl Oligopeptidase. In: Walls, D., Loughran, S. (eds) Protein Chromatography. Methods in Molecular Biology, vol 681. Humana Press. https://doi.org/10.1007/978-1-60761-913-0_12
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DOI: https://doi.org/10.1007/978-1-60761-913-0_12
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